Jan 02, 2025
Organoid well plate scanning
- Joe Airey1
- 1Nikon healthcare UK
- Nikon Healthcare UK, Branch of Nikon Europe
Protocol Citation: Joe Airey 2025. Organoid well plate scanning . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr98epvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working however customization of the channels, thresholding settings, and optical configurations will be specific for each individual use case.
Created: December 10, 2024
Last Modified: January 02, 2025
Protocol Integer ID: 114658
Keywords: organoid, well plate, automated, Nikon, NIS elements, Z-stack, Z stack, spheroid , stack positions for each organoid, plate at 4x magnification, imaging routine, xy position of organoid, imaging, stack at 20x magnification, 4x magnification, 20x magnification, nis elements software, jobs module within nikon, nikon, xy position, stack position, plate
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Abstract
This is a conditional or 'smart' imaging routine written using the JOBs module within Nikon's NIS elements software (version 6.02.03). The JOB is designed to serially scan each well within a well-plate at 4x magnification and identify the XY position of organoids in each well. The program will then loop over those XY positions, identifying the top and bottom Z-stack positions for each organoid and then perform a Z-stack at 20x magnification.
Materials
Z_abs_bounds_20x.ga3269KB 
Z_abs_bounds_4x.ga3272KB 
Organoid_wellplate_3.bin70KB Required:
4x air objective
20x air objective
Nikon Ti2E microscope with motorized nosepiece and stage
96 well-plate
Attached is a NIS elements JOB designed to:
1) Scan over a 96 well-plate using the 4x objective and identify the XY coordinates of organoids in each well
2) Return to the XY coordinates of identified organoids and switch to the 20x air objective
3) Perform a coarse Z stack with a large step size at 20x magnification
4)Threshold the coarse Z stack to automatically segment and identify the top and bottom of the organoid
5) Perform a high resolution Z stack at 20x using the Z dimensions from the segmentation
Troubleshooting
Manual focusing on the first well
Create optical configurations (OCs) for the fluorophores you wish to image. For this JOB you will need an OC linked to a 4x objective and and OC linked to a 20x air objective.
Using the joystick move the stage so that the objective is beneath the first well to be imaged (usually top left corner of the well plate).
'Go live' and focus on the organoid using the 4x objective. Focus on the brightest point of the organoid.
Importing and running the JOB
Search 'JOBs explorer' in the search bar in the top right corner of NIS elements.
Import the JOB into the JOBs explorer
Select the JOB named 'Organoid_wellplate_3'
Run the JOB by clicking the button that looks like the 'play' symbol. This will open the JOB setup wizard where you can select imaging parameters.
Import the required general analysis 3 (GA3) recipes that work within the JOB to segment and save the XYZ locations of the organoids. You can do this by double clicking on the GA3 command in the JOB highlighted in red in the image below and then importing the GA3 recipes. The upper GA3 command should have the 'Z_abs_bounds_4x' GA3 recipe imported, and the lower GA3 command should have the 'Z_abs_bounds_20x' GA3 recipe imported.
Select the step size you would like to use for the high resolution Z-stacks. This is at the top of the setup wizard in the field named: 'Z Step Size?' and takes an integer.
Select the high magnification capture definition. This is where you'll input the 20x magnification optical configurations (OC) you made earlier.
Select the low magnification capture definition. This is where you'll input the 4x magnification optical configurations (OC) you made earlier.
Define the depth (in Z) and step size (in micrometers) you would like the microscope to search over when looking for organoids in each well. This is by default 500um range with 10um steps.
Select which wells you would like to image by clicking and dragging across the well area
Click 'Run' !